Our laboratories have a long-standing interest in immune tolerance in transplantation and autoimmunity, with a particular focus on regulatory T cells (Tregs). The primary population of Tregs in humans and mice is defined by expression of the X-linked transcription factor Foxp3. While these cells are required for normal immune homeostasis increasing data suggests that this lineage of cells may be unstable (e.g., as a result for example of inadequate IL-2, TCR and CD28 stimulation, or due to exposure to inflammatory cytokines), meaning that Tregs can revert/convert to effector T cells and thus contribute to loss of tolerance to self or to transplanted allografts. One of the key pathways controlling lymphocyte lineage specification and responsiveness is the phosphoinositide 3-kinase (PI3K) pathway, which can be activated via multiple surface receptors, including, most prominently, CD28 and the IL-2R. While this pathway is essential for conventional T cell responses, it may have limited, if any, function in Tregs. In fact, over activation of the PI3K pathway dramatically inhibits Treg development, while the STAT5 pathway, which is also activated through the IL-2R, strongly promotes Tregs. The primary regulator of PI3K activity in T cells is the lipid phosphatase PTEN (phosphatase and tensin homolog on chromosome 10). The goal of this proposal is to determine how PTEN in Tregs controls Treg homeostasis, the integration of PI3K and STAT5 signals, and whether drug targeting of the PI3K pathway can stabilize Tregs. To accomplish this, we created mice with PTEN deleted specifically in Tregs by breeding mice with a PTENfl/fl allele with Foxp3-YFP-Cre knock-in or BAC transgenic animals to generate PTEN-Treg mice. Surprisingly, although these mice have elevated numbers of Tregs, a high proportion of those cells are CD25- and CD62Llo, and the animals develop a severe polyclonal lymphoproliferative disorder. This has led us to formulate the hypothesis that PTEN loss disrupts Treg homeostasis due to reduced cytokine receptor expression, altered migration and apparent loss of regulatory capacity. The goal of this grant is to determine how PTEN in Tregs controls Treg homeostasis, the integration of PI3K and STAT5 signals, and whether drug targeting of the PI3K pathway can stabilize Tregs. To do so, we have two aims. In Aim #1, employing Treg fate mapping mice with Treg specific deletion of PTEN, we will examine the effects of loss of PTEN on natural and adaptive Treg stability and function, both under homeostatic conditions and in a model autoimmune disease. In Aim #2, we will dissect the signals downstream of CD25, PI3K and STAT5 to determine which are responsible for the Treg phenotypic and functional changes we have observed. Our studies will yield new insights into Treg signaling pathways and provide potential therapeutic strategies to enhance immune tolerance.